CN106290205B - Application of the measuring method of glycolipid content in wheat flour in a kind of flour - Google Patents

Application of the measuring method of glycolipid content in wheat flour in a kind of flour Download PDF

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CN106290205B
CN106290205B CN201610997440.8A CN201610997440A CN106290205B CN 106290205 B CN106290205 B CN 106290205B CN 201610997440 A CN201610997440 A CN 201610997440A CN 106290205 B CN106290205 B CN 106290205B
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dialycerides
content
galactolipin
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double
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马冬云
秦海霞
侯俊峰
黄鑫
王晨阳
杨国玉
韩巧霞
郭天财
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Henan Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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    • G—PHYSICS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder

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Abstract

The present invention relates to the measuring method and its application of glycolipid content in a kind of flour, which includes the following steps：（1）Using the total sugar content of respective components in colorimetric method for determining flour；（2）In conjunction with step（1）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates the content of double galactolipin dialycerides, and wherein x is total sugar content, y1For double galactolipin dialycerides contents；In conjunction with step（1）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates the content of single galactolipin dialycerides, and wherein x is total sugar content, y2Single galactolipin dialycerides content.This method measurement is easy, quick, can accurately measure polar glycolipids content in flour or starch.

Description

Application of the measuring method of glycolipid content in wheat flour in a kind of flour
Technical field
The invention belongs to food technology fields, and in particular to the measuring method of glycolipid content and its answer into a kind of flour With.
Background technique
Plant galactolipid, which refers to, is widely present in the polarity lipid containing galactose residue in plant.Galactolipid is wide It is general to be distributed in plant kingdom, it is most important glycolipid ingredient in plant tissue, main component is single galactolipid and double galactolipins Rouge.Containing the lipid of 0.5%-3% in flour, wherein glycolipid accounts for 26.4%.And single galactolipin dialycerides and double galactolipin glycerol two Rouge accounts for the 69% of glycolipid again.Polarity lipid can improve the persistence of dough in flour, play good action to loaf volume is increased.Rouge Class and carbohydrate are all present in wheat seed, their mutual work plays an important role to the baking properties of food.Lipid The rheological properties of starch gel are affected, the formation of crumb texture structure is delayed, extend bread Bulking Time.Meanwhile Polar lipid, especially starch surface polarity rouge and the close phase of grain hardness in flour are found in the hardness research to wheat seed It closes.Its lipoid bond area soft textured protein Friabilin is one and is easy to combine rich in triptophan domain and starch surface, To influence wheat hardness.Film fixation ring is formed between tryptophan area d- spiral to combine closely with starch surface bimolecular polar lipid, Soft wheat starch dissimulated electricity state polar lipid (glycolipid and phosphatide) content is apparently higher than hard wheat.
In terms of galactolipid quantitative analysis, majority is to first pass through column chromatography to isolate and purify to obtain half with thin layer analysis (TLC) Then lactose rouge carries out methylation sour water solution, gas-liquid chromatography technology is recycled to analyse fatty acid methyl ester, is finally converted into half The content of lactose rouge.But cumbersome, technical requirements are high, and as a result poor reproducibility, success rate are low.Also useful high resolution mass spectrum ESI- QTOF/MS carrys out quantitative analysis, but instrument used in this method is more expensive, cannot in high volume and under the conditions of a wide range of answer With.Contain a large amount of polyunsaturated fatty acid using galactolipid in addition, also having, has light absorption height at 200nm wavelength or so place The characteristics of peak, uses reversed-phased high performace liquid chromatographic（HPLC it) is measured to analyze；This measuring method is efficient and convenient, but cost of equipment Range that is higher, limiting its application.It is also it has been proposed that straight in order to quickly and easily measure the glycolipid content in flour and starch The method of measurement total sugar content is connect to estimate its glycolipid content, but this method is inaccurate, error is larger.Therefore, one is established Kind is accurate, the quick and at low cost method for measuring glycolipid content in flour and starch is very necessary.
Summary of the invention
It is an object of that present invention to provide the measuring method and its application of glycolipid content in a kind of flour, this method can be quick Accurately measure polar glycolipids content in flour or starch.
In order to achieve the above object, the technical solution adopted by the present invention is that：
The present invention provides a kind of measuring methods of glycolipid content in flour, include the following steps：
（1）Using the total sugar content of respective components in colorimetric method for determining flour；
（2）In conjunction with step（1）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double galas The content of sugared dialycerides, wherein x is total sugar content, y1For double galactolipin dialycerides contents；In conjunction with step（1）Obtained in Total sugar content, according to equation y2=2.3567x-1.2032 calculates the content of single galactolipin dialycerides, and wherein x contains for total reducing sugar Amount, y2Single galactolipin dialycerides content.
Application of the measuring method of glycolipid content in wheat flour in above-mentioned flour, specific step is as follows：
（1）First stirring dipping in water saturated butanol solution is added to obtain mixture a, wheat the wheat flour of 40 ~ 80 mesh The mass ratio of powder and water saturated butanol solution is 1:（6~7）, dip time is 1 ~ 2h；Then by mixture a in temperature 30 ~ 48 ~ 72 h of oscillation extraction, are then centrifuged 15 ~ 20 min in 3000 ~ 4000 rpm, obtain the supernatant of yellow transparent at 40 DEG C；
（2）By step（1）Obtained supernatant is concentrated into 4 ~ 5mL in 35 ~ 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 ~ 4 times with extractant c, and it is molten to merge lower phase layer yellow Liquid obtains mixed liquor d, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 ~ 2.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 ~ 3 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 0.8 ~ 2mL methanol, obtains thick rouge；
（8）Take 200 ~ 500 μ L steps（7）Obtained in thick rouge, the distance bottom on the silica gel plate of specification 200mm * 200mm Point sample at 1 ~ 3cm of portion, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume Than being 10:2:2:2:1 configures；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, found out respectively Band where purpose object list galactolipin dialycerides and double galactolipin dialycerides, digs down；
（10）The band where purpose object is placed in centrifuge tube respectively, it is molten at 40 DEG C of temperature with 10 ~ 20mL lysate g Solve 5 ~ 10min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in title In centrifuge tube after weight；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of purpose object is calculated separately out with difference assay；
（14）Purpose object is dissolved separately in 1 M KOH- ethanol solution, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:（1~2）, 20 ~ 30 min then are kept the temperature in temperature 70 C, 3 ~ 4mL, 1 mol/L hydrochloric acid are then added, then in temperature 99 DEG C of 15 min of heat preservation are spent, 7.5 ~ 14mL 1mol/L KOH is eventually adding；
（15）The step of pipetting 2 ~ 6ml（14）The supernatant in solution is obtained to be placed in different centrifuge tubes, in temperature 60 ~ It is evaporated at 80 DEG C, distills water washing with 3 ~ 4ml respectively, 3 ~ 4ml Fehling Regent is added into each centrifuge tube, in temperature 99 DEG C heat preservation 10min, be added 4mL sulfuric acid-ammonium molybdate color developing agent, be settled to 10 mL respectively；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates total sugar content in different centrifuge tubes according to galactolipin graticule；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is total sugar content, y1For double galactolipin dialycerides contents；In conjunction with step（17）In The total sugar content arrived, according to equation y2=2.3567x-1.2032 calculates the content of single galactolipin dialycerides, and wherein x is total Sugared content, y2Single galactolipin dialycerides content.
The present invention is with advantage is compared with other methods：
The method of the present invention measurement is easy, quick, accurate, provides a kind of easy side for fast and accurately measuring glycolipid content Method.
Specific embodiment
Embodiment 1
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 ~ 80 mesh, the 10.000 g wheat breed day people, 198 flour is added in the water saturated butanol solution of 60mL Stirring dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated to 48 h of extraction at 35 DEG C of temperature, then 15 min are centrifuged in 4000 rpm, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 35 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, found out respectively Band where purpose object list galactolipin dialycerides and double galactolipin dialycerides, digs down；
（10）Band where purpose object is respectively placed in different centrifuge tubes, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated separately out with difference assay；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step respectively（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, distills water washing with 3 ml, to 3ml Fehling Regent is added in centrifuge tube, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in its people 198 The sugared content of sugared dialycerides is 3.040 mg/g, and the sugared content of single galactolipin dialycerides is 0.7483 mg/g；
（18）In conjunction with step（17）Obtained in different component total sugar content, according to equation y1=0.0271x-0.0288 meter The content of double galactolipin dialycerides is calculated, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin glycerol two Rouge content；In conjunction with step（17）Obtained in total sugar content, according to equation y2It is sweet that=2.3567x-1.2032 calculates single galactolipin The content of oily two rouge, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；According to formula The content for calculating double galactolipin dialycerides in day people 198 is 0.0536mg/g, and the content of single galactolipin dialycerides is 0.5603 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10 uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides；Double galactolipin dialycerides in its people 198 Content be 0.0588 mg/g, the contents of single galactolipin dialycerides is 0.6005 mg/g.
Embodiment 2
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed week wheat, 32 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in all wheats 32 The sugared content of sugared dialycerides is 2.271mg/g, and the sugared content of single galactolipin dialycerides is 0.8617 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in all wheats 32 The content of sugared dialycerides is 0.0327 mg/g, and the content of single lactose dialycerides is 0.8276 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in all wheats 32 Content is 0.0301mg/g, and the content of single galactolipin dialycerides is 0.8215 mg/g.
Embodiment 3
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed, hundred agriculture, 207 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in centrifuge tube respectively, is dissolved at 40 DEG C of temperature with 15mL lysate g It according to volume ratio is 2 that 5min, lysate g, which are by chloroform and methanol,:1 configures, and then filters, after filtrate is placed in weighing In centrifuge tube；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated separately out with difference assay；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10 mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in hundred agricultures 207 The sugared content of sugared dialycerides is 2.234mg/g, and the sugared content of single galactolipin dialycerides is 0.7937 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in hundred agricultures 207 The content of sugared dialycerides is 0.0317 mg/g, and the content of single lactose dialycerides is 0.6673 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18 (250mm × 4.6mm), under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides；Double galactolipin dialycerides in hundred agricultures 207 Content be 0.0204 mg/g, the contents of single galactolipin dialycerides is 0.6508 mg/g.
Embodiment 4
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）40 mesh, 10.000 g wheat breed Feng De is first deposited into No. 1 flour add in the water saturated butanol solution of 60mL and stirs It mixes dipping and obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Feng De is deposited double half in No. 1 The sugared content of lactose dialycerides is 2.088mg/g, and the sugared content of single galactolipin dialycerides is 0.8390mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Feng De is deposited double half in No. 1 The content of lactose dialycerides is 0.0278mg/g, and the content of single lactose dialycerides is 0.7741mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Feng De deposits double galactolipin glycerol two in No. 1 The content of rouge is 0.0263mg/g, and the content of single galactolipin dialycerides is 0.7821 mg/g.
Embodiment 5
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed Zheng wheat, 21 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in Zheng wheat 21 The sugared content of sugared dialycerides is 2.271mg/g, and the sugared content of single galactolipin dialycerides is 0.5669mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in Zheng wheat 21 The content of sugared dialycerides is 0.0327 mg/g, and the content of single lactose dialycerides is 0.1328 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in Zheng wheat 21 Content is 0.0310mg/g, and the content of single galactolipin dialycerides is 0.1203 mg/g.
Embodiment 6
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed Zheng wheat, 113 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galactolipins in Zheng 113 The sugared content of dialycerides is 2.821mg/g, and the sugared content of single galactolipin dialycerides is 0.839 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in Zheng wheat 113 The content of sugared dialycerides is 0.0476mg/g, and the content of single lactose dialycerides is 0.7741mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in Zheng 113 Content is 0.0443mg/g, and the content of single galactolipin dialycerides is 0.7601 mg/g.
Embodiment 7
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First expensive 257 flour of agriculture of 40 mesh, 10.000 g wheat breed is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in your agriculture 257 The sugared content of sugared dialycerides is 2.564mg/g, and the sugared content of single galactolipin dialycerides is 0.802 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in your agriculture 257 The content of sugared dialycerides is 0.0407 mg/g, and the content of single lactose dialycerides is 0.6869 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in your agriculture 257 Content be 0.0431mg/g, the contents of single galactolipin dialycerides is 0.7022mg/g.
Embodiment 8
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed Yangmai No.158 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in Yangmai No.158 The sugared content of sugared dialycerides is 2.125mg/g, and the sugared content of single galactolipin dialycerides is 0.615mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in Yangmai No.158 The content of sugared dialycerides is 0.0288mg/g, and the content of single lactose dialycerides is 0.247mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in Yangmai No.158 Content be 0.0306mg/g, the contents of single galactolipin dialycerides is 0.225 mg/g.
Embodiment 9
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 40 mesh, 10.000 g wheat breed silk floss wheat, 11 flour is added in the water saturated butanol solution of 60mL and is stirred Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；Double galas in continuous wheat 11 The sugared content of sugared dialycerides is 2.967mg/g, and the sugared content of single galactolipin dialycerides is 0.8163 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）Obtained in total sugar content, according to equation y2=2.3567x-1.2032 calculates single galactolipin dialycerides Content, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；Double galas in continuous wheat 11 The content of sugared dialycerides is 0.0516mg/g, and the content of single lactose dialycerides is 0.7206 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18 (250mm × 4.6mm), under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.Double galactolipin dialycerides in continuous wheat 11 Content is 0.0608mg/g, and the content of single galactolipin dialycerides is 0.7115 mg/g.
Embodiment 10
Application of the measuring method of glycolipid content in wheat flour in a kind of flour, specific step is as follows：
（1）First 12 flour being educated in 40 mesh, 10.000 g wheat breed and adding in the water saturated butanol solution of 60mL stir Dipping obtains mixture a, dip time 1.5h；Then mixture a is vibrated at 35 DEG C of temperature extraction 48 h, then in 4000 rpm are centrifuged 15 min, obtain the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 5mL in 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, extractant c be by chloroform, first alcohol and water by It is 2 according to volume ratio:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, The mixed liquor of upper phase layer then uses extractant c to be extracted, and repeats to be operated 2 times with extractant c, merges lower phase layer yellow solution Mixed liquor d is obtained, then dries up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 1.6mL methanol, obtains thick rouge；
（8）Take 400 μ L steps（7）Obtained in thick rouge, apart from bottom on the silica gel plate of specification 200mm * 200mm Point sample at 2cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；At 0.5 cm of chromatoplate edge, stop chromatography, takes out thin layer chromatography board and be placed on experiment On platform, dried up with hair dryer；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out single half Band where lactose dialycerides and double galactolipin dialycerides purpose objects, digs down；
（10）The band where purpose object is placed in different centrifuge tubes respectively, with 15mL lysate g at 40 DEG C of temperature Dissolve 5min, it according to volume ratio is 2 that lysate g, which is by chloroform and methanol,:1 configures, and then filters, and filtrate is placed in weighing In centrifuge tube afterwards；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The total weight of different purpose objects is calculated with difference assay respectively；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the matter of purpose object and 1 M KOH- ethanol solution Amount is than being 1:2,20 ~ 30 min then are kept the temperature in temperature 70 C, 1 mol/L hydrochloric acid of 3mL are then added, then in 99 DEG C of temperature 15 min are kept the temperature, 7.5mL 1mol/L KOH aqueous solution is eventually adding；
（15）Removing step（14）Middle solution 2ml supernatant, is evaporated at 80 DEG C, water washing is distilled with 3 ml, to centrifugation 3ml Fehling Regent is added in pipe, 99 DEG C of 10 min of water-bath are added 4 ml sulfuric acid-ammonium molybdate color developing agent, are settled to 10mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates sugared content according to galactolipin graticule；In educate in 12 double galas The sugared content of sugared dialycerides is 2.564mg/g, and the sugared content of single galactolipin dialycerides is 0.7937 mg/g；
（18）In conjunction with step（17）Obtained in total sugar content, according to equation y1=0.0271x-0.0288 calculates double half The content of lactose dialycerides, wherein x is the sugared content of double galactolipin dialycerides, y1For double galactolipin dialycerides contents； In conjunction with step（17）In 0.7937 obtained total sugar content, according to equation y2It is sweet that=2.3567x-1.2032 calculates single galactolipin The content of oily two rouge, wherein x is the sugared content of single galactolipin dialycerides, y2Single galactolipin dialycerides content；In educate in 12 The content of double galactolipin dialycerides is 0.0407 mg/g, and the content of single lactose dialycerides is 0.6672 mg/g；
（19）Repeat step（1）It arrives（11）, by step（11）Obtained in filtrate with being dried with nitrogen；
（20）It is dissolved in substance is obtained in step 19 in 800 ul methanol, is 205 nm, mobile phase 95% in Detection wavelength Methanol, 5% 0.2% acetic acid solvent system, flow velocity 1mL/min, column temperature is room temperature, Symmetry c18（250mm× 4.6mm）, under conditions of sample volume is 10uL, efficient liquid phase measurement is carried out to sample, carries out quantitative analysis with external standard method, respectively Obtain the content of polar lipid list galactolipin dialycerides and double galactolipin dialycerides.In educate in 12 double galactolipin dialycerides Content is 0.0361mg/g, and the content of single galactolipin dialycerides is 0.6865 mg/g.
Single galactolipin dialycerides calculated value and actual measured value Comparative result of 1 this method of table
Double galactolipin dialycerides calculated values and actual measured value Comparative result of 2 this method of table

Claims (1)

1. application of the measuring method of glycolipid content in wheat flour, includes the following steps in a kind of flour：
1）Using the total sugar content of the respective components in colorimetric method for determining flour：
（1）First add in water saturated butanol solution stirring dipping to obtain mixture a the wheat flour of 40 ~ 80 mesh, wheat flour with The mass ratio of water saturated butanol solution is 1:（6~7）, dip time is 1 ~ 2h；Then by mixture a in 30 ~ 40 DEG C of temperature Lower oscillation extracts 48 ~ 72 h, is then centrifuged 15 ~ 20 min in 3000 ~ 4000 rpm, obtains the supernatant of yellow transparent；
（2）By step（1）Obtained supernatant is concentrated into 4 ~ 5mL in 35 ~ 40 DEG C of temperature, obtains concentrate b；
（3）By step（2）Obtained concentrate b is extracted with extractant c, and extractant c is by chloroform, first alcohol and water according to body Product is than being 2:1:0.75 mixed configuration forms, and phase layer and lower phase layer are divided into after extraction, and it is spare to remove phase layer yellow solution；
（4）Step（3）In the mixed liquor of upper phase layer then use extractant c to carry out extraction split-phase, lower phase layer yellow solution, upper phase The mixed liquor of layer then uses extractant c to be extracted, and repeats to be operated 2 ~ 4 times with extractant c, merges lower phase layer yellow solution and obtains To mixed liquor d, then dry up mixed liquor d to obtain Mischung using nitrogen；
（5）It takes 1.5 ~ 2.5 mL chloroformic solutions to wash Mischung, is dried with nitrogen；
（6）Repeat step（5）Operation 2 ~ 3 times, obtains substance f；
（7）By step（6）Obtained substance f, which is dissolved in 0.8 ~ 2mL methanol, obtains thick rouge；
（8）Take 200 ~ 500 μ L steps（7）Obtained in thick rouge, on the silica gel plate of specification 200mm * 200mm apart from bottom 1 ~ Point sample at 3cm, panel under the action of solvent, solvent are by chloroform：Methanol：Glacial acetic acid：Acetone：Water is according to volume ratio 10:2:2:2:1 configures；
（9）Different spots can be seen under 254 nm wavelength illumination of ultraviolet lamp, under the control of standard items, find out purpose respectively Band where object list galactolipin dialycerides and double galactolipin dialycerides, digs down；
（10）Band where purpose object is respectively placed in different centrifuge tubes, respectively with 10 ~ 20mL lysate g in 40 DEG C of temperature It according to volume ratio is 2 that lower dissolution 5 ~ 10min, lysate g, which are by chloroform and methanol,:1 configures, and then filters, and filtrate is set In different centrifuge tubes after weighing；
（11）Repeat step（10）, then merging filtrate respectively；
（12）By step（11）Obtained in filtrate with being dried with nitrogen, weigh to centrifuge tube；
（13）The weight of purpose object list galactolipin dialycerides and double galactolipin dialycerides is calculated separately out with difference assay；
（14）Purpose object is dissolved in 1 M KOH- ethanol solution respectively, the mass ratio of purpose object and 1 M KOH- ethanol solution It is 1:（1~2）, 20 ~ 30 min then are kept the temperature in temperature 70 C, 3 ~ 4mL, 1 mol/L hydrochloric acid are then added, then in temperature 99 DEG C heat preservation 15 min, be eventually adding 7.5 ~ 14mL 1mol/L KOH；
（15）2 ~ 6ml step is pipetted respectively（14）It obtains the supernatant in solution to be placed in different centrifuge tubes, in temperature 60 ~ 80 It is evaporated at DEG C, distills water washing with 3 ~ 4ml, 3 ~ 4ml Fehling Regent is added into centrifuge tube and adds in 99 DEG C of heat preservation 10min of temperature Enter 4mL sulfuric acid-ammonium molybdate color developing agent, is settled to 10 mL；
（16）By step（15）Solution measure light absorption value under 600nm wavelength respectively；
（17）According to step（16）The light absorption value of measurement calculates total sugar content in different centrifuge tubes according to galactolipin graticule；
2）In conjunction with step 1）Obtained in each component total sugar content, according to equation y1=0.0271x-0.0288 calculates double galactolipins The content of dialycerides, wherein x is total sugar content, y1For double galactolipin dialycerides contents；In conjunction with step 1）Obtained in it is each Component total sugar content, according to equation y2=2.3567x-1.2032 calculates the content of single galactolipin dialycerides, and wherein x is total Sugared content, y2Single galactolipin dialycerides content；The content unit is mg/g.
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